HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening.

Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver-specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg-Gly-Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening.